This proposal will extend the studies of the parent award to investigate the regulation of tau liquid liquid phase separation (LLPS) and fibrillization by RNA binding proteins (RBPs). The field has discovered that RNA binding proteins and tau protein form RNA granules (membraneless organelles) through a process of liquid liquid phase separation (LLPS). This process seems to be important for formation of stress granules, and it is hypothesized to be important for Alzheimer?s disease and tauopathy. While investigating LLPS of tau we discovered that the interaction of tau with RBPs, including Tia1 and G3BP, controls the pathway of tau aggregation, with dramatic results. We find that the type of RNA binding protein interacting with tau causes dramatic shifts in its behavior. For instance, mixing tau with Tia1 causes tau to initially phase separate with Tia1, but then over time coalesce within the Tia1 droplets to form micro-aggregates, while tau remains dispersed in LLPS droplets after mixing tau with G3BP. These observations parallel the results that we observe in cells and in vivo, where tau pathology associates with Tia1, but not with G3BP. These results point towards a major expansion in our understanding of the pathophysiology of tauopathy because they suggest that RBPs stimulate pathological tau aggregation, and might explain why particular RBPs are associated with tau pathology. The work in this application will investigate how RBPs control of phase separation, and determine whether particular RBPs bias tau interactions towards pathological aggregation and fibrillization. We hypothesize that the interaction between tau, RNA binding proteins and RNA controls the aggregation behavior of tau (phase separation vs. fibrillization). In Aim 1, we will determine the biophysical conditions regulating Tau Phase Separation vs. Fibrillization Pathways by Tia1, and compare to G3BP1 and hnRNPA1. In Aim 2 we will determine whether Tia1 controls Tau Phase Separation vs. Fibrillization Pathways in primary cell cultures of cortical neurons. We expect these experiments to lead to a major advance in our understanding of the control of physiological and pathological tau LLPS.